Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

Complete inhibition of purinoceptor agonist-induced nociception by spinorphin, but not by morphine

We found that spinorphin, a novel neuropeptide showed analgesia in a different manner compared with morphine. By measuring flexor responses induced by the intraplanter injection of substances, the presence of three different types of sensory neurons were demonstrated. Although spinorphin completely blocked 2-metylthioadenosine (2-MeS ATP, a P2X(3) agonist)-induced responses, morphine did not. On the other hand, morphine-induced blockade of bradykinin (BK, a B(2)-receptor agonist)-responses was attenuated by pertussis toxin (PTX) treatment, whereas that of spinorphin was not. Thus it is suggested that spinorphin has a spectrum of analgesia which covers the blockade of nociception insensitive to morphine.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.     

A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains

Maximum growth for Rhizopus sp. A-11 was obtained at a zinc ion concentration of 0.7 ppm in a liquid medium. Glucoamylase (GA, EC 3.2.1.3) production in Rhizopus sp. A-11 was maximized at 710 U/ml, at the presence of 75 ppm for calcium and 0.7 ppm of zinc ions in liquid medium. Zinc ion is known as an essential biometal for Rhizopus growth; however, growth was inhibited by the zinc ion concentration, not maximized. Although calcium ion was not necessary to Rhizopus growth, GA production using Rhizopus sp. A-11 was markedly stimulated by calcium ion concentration over 75 ppm in the liquid medium. The GA productivity of the present liquid culture was about 4.4 times higher than that of the solid state culture, based on the unit starch amount in the liquid and solid media carbon source. The characteristics of the GA produced by the Rhizopus sp. A-11 liquid culture were interesting; that is, almost all the GA produced was classified as raw starch-digesting GA (GA-I). Secreted protein in the culture liquid after 30 h was nearly GA, and had a limited amount of impure protein. As a result, it was found that using a Rhizopus culture in a specified metal-ion regulated medium was an effective method for producing GA. Thus the present culture method was renamed the "metal-ion-regulated liquid culture method".     

Identification of 4-Trimethylaminobutyraldehyde Dehydrogenase (TMABA-DH) as a Candidate Serum Autoantibody Target for Kawasaki Disease

Kawasaki disease (KD), an acute vasculitis that preferentially affects coronary arteries, is still the leading cause of acquired heart disease in children. Although the involvement of immune system malfunction in the onset of KD is suggested, its etiology still remains to be clarified. We investigated autoantibodies in KD patients, which are frequently found in sera from patients with autoimmune diseases, vasculitides and arteritides. We performed two-dimensional western blotting and LC-MS/MS to analyze the antigens of autoantibodies, detected two protein spots with 4 out of 24 sera from KD patients but not with 6 control sera, and identified the antigens as 4-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH). A slot blot analysis with TMABA-DH as an antigen also revealed higher reactivities of patients'' sera than control sera (positive rates: 18/43 vs 3/41). Using an enzyme-linked immunosorbent assay (ELISA), we found that the reactivity of anti-TMABA-DH antibodies in sera from KD patients was significantly higher than that in sera from age-matched controls. The optimal cut-off value of 0.043 had a sensitivity of 83.7% and a specificity of 80.0% in detecting KD patients (positive rates: 37/43 for KD patients, 9/41 for controls). Immunohistochemistry performed on thin sections of rat heart revealed that TMABA-DH colocalized with myosin light chains in cardiac myocytes. Patient sera with high reactivity gave similar immunostaining pattern. These results suggest that the detection of anti-TMABA-DH autoantibody could be a potential strategy for a diagnosis of KD.     

Role of Macrophage Migration Inhibitory Factor (MIF) in Pollen-Induced Allergic Conjunctivitis and Pollen Dermatitis in Mice

Pollen is a clinically important airborne allergen and one of the major causes of allergic conjunctivitis. A subpopulation of patients with atopic dermatitis (AD) are also known to have exacerbated skin eruptions on the face, especially around the eyelids, after contact with pollen. This pollen-induced skin reaction is now known as pollen dermatitis. Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that plays an essential role in allergic inflammation. Recent findings suggest that MIF is involved in several allergic disorders, including AD. In this study, MIF knockout (KO), MIF transgenic (Tg) and WT littermate mice were immunized with ragweed (RW) pollen or Japanese cedar (JC) pollen and challenged via eye drops. We observed that the numbers of conjunctiva- and eyelid-infiltrating eosinophils were significantly increased in RW and JC pollen-sensitized MIF Tg compared with WT mice or MIF KO mice. The mRNA expression levels of eotaxin, interleukin (IL)-5 and IL-13 were increased in pollen-sensitized eyelid skin sites of MIF Tg mice. An in vitro analysis revealed that high eotaxin expression was induced in dermal fibroblasts by MIF combined with stimulation of IL-4 or IL-13. This eotaxin expression was inhibited by the treatment with CD74 siRNA in fibroblasts. These findings indicate that MIF can induce eosinophil accumulation in the conjunctiva and eyelid dermis exposed to pollen. Therefore, targeted inhibition of MIF might result as a new option to control pollen-induced allergic conjunctivitis and pollen dermatitis.